In a chemically defined medium with glucose and sucrose as major carbon sources (standard medium), Cephalosporium acremonium excretes the intermediate of the β-lactam biosynthetic pathway, penicillin N, into the medium during growth; production of cephalosporins is delayed until glucose is completely utilized. Deacetoxycephalosporin C synthetase, the ring-expansion enzyme (“expandase”), does not appear as long as glucose is present. Afterwards, initiation of its formation is accompanied by the production of cephalosporins. Feeding additional glucose during the fermentation turns off expandase synthesis without affecting formation of isopenicillin N synthetase, the ring-cyclization enzyme (“cyclase”). The above results point to a strong glucose catabolite repression of the expandase as one of the main regulatory mechanisms in β-lactam biosynthesis by Cephalosporium acremonium and the reason for accumulation of penicillin N during the fermentation. Cyclase shows a biphasic pattern in activity, the first very high peak not being correlated with the excretion of any β-lactam antibiotic into the medium.